Methods for directly binding antigens or antibodies to be detected to a solid support are well known. Also known are methods of indirectly binding an antigen or antibody to be detected to a solid support by first coating the solid support with the binding partner of the species to be detected. Generally, the solid support having the antigen or antibody to be detected bound thereto is further reacted with labeled (radioactive, enzyme, fluorescent, stable-free radical, etc.) binding partner of the antigen or antibody bound to the solid support and the amount of label bound to the solid support is determined.
Haptens are protein free bodies, generally of low molecular weight that do not induce antibody formation when injected into an animal, but are reactive to antibodies.
Antibodies to hapten are raised by first conjugating the hapten to a protein and injecting the conjugated product into an animal or human. The resulting antibodies are isolated by conventional antibody isolation techniques. Examples of haptens are steroids such as esterone, estrodiol, testosterone and progesterone. Vitamins such as vitamin B-12 and folic acid, thyroxine, thyrodothyroxine, histamine, serotonis, digoxin, prostaglandin, adrenalin, noradenalin, kinetin, gibberellic acid and 2,4-dinitrophenol are suitable hapten molecules.
The 2,4-dinitrophenyl (DNP) moiety is particularly useful hapten antigenic site. This group is conveniently introduced by reacting 2,4-dinitrophenol, 2,4-dinitroaniline, or .epsilon.-DNP-L-lysine with the antibody for the antibody or antigen to be detected.
Those skilled in the immunoassay arts will recognize a wide variety of methods for binding antibodies or antigens to solid supports; U.S. Pat. Nos. 3,939,350; 3,853,987; 3,654,090; 3,646,346 and 3,867,517. Techniques for labeling antibodies with iodine-125 (.sup.125 I) or other radioactive labels are well known: Greenwood, Hunter and Glover, Biochem. J., 89:114, (1963).
Techniques for fluorescently labeling antibodies are also well known: Feltkamp, Immunology 18, 875 (1970) and U.S. Pat. No. 3,789,116.
Likewise, enzymes such as catalase, peroxidase .beta.-glucouronidase, glucose-6-phosphate dyhydrogenase, urease, and glucoseoxidase are conveniently linked to antibodies by art recognized techniques: U.S. Pat. Nos. 3,875,011; 3,791,932 and 3,879,262.